At present the virulence properties enabling Escherichia coli to act as one of the major opportunistic pathogens are not well understood. E. coli hemolysin (hly) plays a contributory role in the virulence of extraintestinal infections. We isolated a chromosomal hly determinant in the form of a recombinant plasmid (pSF4000) and localized the hly determinant to a continuous 7 kilobase (kb) portion of pSF4000. By use of current genetic and recombinant DNA methodologies the operon structure of the hly determinant will be characterized. Pulse-chase experiments employing minicell polypeptide analysis and membrane isolation will be used to study the processing and secretion of the E. coli gly. Comparative DNA sequence analysis will be performed on similar 400 basepair (bp) regions of two hly determinants. Differences in hly expression and virulence are localized to this region and the DNA sequence information will eventually lead to understanding how regulation of a virulence factor influences its contribution to pathogenicity. The DNA sequences surrounding several hly determinants will be isolated as recombinant cosmid-type plasmids and examined for their ability to encode other virulence properties. DNA-DNA hybridization experiments utilizing the Southern blotting technique will enable examination of the DNA sequence environment surrounding the chromosomal hly determinant of different clinical isolates of E. coli. These experiments will identify the physical unit involved in the complete horizontal transmission of the hly determinant in E. coli. Based on experiments using a rat model of E. coli peritonitis, haptoglobin is proposed to have therapeutic potential in hemoglobin or blood enhanced infections. Hemolytic and nonhemolytic isogenic strains of E. coli will be used in the peritonitis model and the protective value of haptoglobin assessed. An important objective of this research is to eventually elucidate how hly acts as a virulence factor. Overall, the long-term goal of this research program is to identify and characterize the genetic and physiological nature of bacterial factors influencing E. coli extraintestinal infections. This type of information is of great value in the development of selective strategies for the prevention and control of serious human bacterial infections.